Introducing foreign gene information in the form of RNA or DNA into cells can be achieved for example by using liposomal and non-liposomal formulations or by physical methods such as electroporation. Although these methods are broadly used for research purposes, pharmaceutical delivery of nucleic acids in vivo remains a challenge as formulations such as liposomes are often found to be unstable and/or toxic.
The present inventor has previously established conditions allowing the production of particles of different sizes (ranging from 50 nm up to micrometers in average) by mixing together protamine and RNA in defined salt conditions/concentrations/protamine-RNA ratios (Rettig L. et al.; WO 2009/144230 A1). The inventor and others have reported that these formulations can be taken up by cells including immune cells and signal though endosome resident receptors, namely Toll Like Receptors (TLRs), particularly TLR-7 and -8, thereby leading to modular immunostimulation (Rettig et al.; Fotin-Mleczek et al.). However, when messenger RNA (mRNA) was used to generate the particles within the adequate mass ratios, i.e. protamine:RNA=1:2 or higher, the mRNA was not expressed (not translated) in the cells that took up particles. In particular, it has been demonstrated by Fotin-Mleczek et al. that “the complexation process itself can inhibit the translation of mRNA and abolish antigen expression”. The authors conclude that “translability and immune stimulating activity are antidromic in protamine:mRNA complexation of different ratios”. They report that a protamine-RNA mass ratio above 1:2 generates immunostimulating particles (stimulation through TLRs) that are translation-incompetent. These results suggest that, due to its high compaction, no functional RNA can be released from protamine particles made at a protamine:RNA ratio above 1:2.
Therefore, there is a need for particles comprising protamine and RNA, in particular particles comprising protamine and RNA at a protamine:RNA ratio above 1:2, which allow the RNA to be efficiently released and to be functional within the cell.